Acute lung injury (ALI) is a devastating disease characterized by alveolar-epithelial barrier disruption and resultant pulmonary edema and hypoxemia. There are currently no effective disease-modifying therapies and supportive care remains the mainstay of medical management. Ultimately, ALI leads to multi-organ dysfunction and/or death in up to 30% of patients. Neutrophils are clearly important for host defense against bacteria, however toxic neutrophil mediators such as reactive oxygen radicals, granule enzymes and neutrophil extracellular traps also contribute to the pathogenesis of lung injury by causing endothelial, parenchymal and alveolar injury. Neutrophils are prime targets for manipulating the inflammatory response and therefore it is critical to understand the molecular mechanisms that guide neutrophil responses. Dr. Clemens' long-term research goal is to understand how intracellular signaling pathways regulate neutrophil activation during acute lung injury and other inflammatory diseases. Calcium is an evolutionarily conserved signaling messenger that is a central component of multiple signaling pathways. In immune cells, increases in cytoplasmic calcium are controlled via store operated calcium entry (SOCE), where calcium release-activated calcium (CRAC) channels allow influx of extracellular calcium when endoplasmic reticulum (ER) stores are depleted. STIM proteins are ER calcium sensors which sense calcium depletion and directly gate ORAI, a pore subunit of the CRAC channel. Little is known about STIM, ORAI or the molecular regulation of calcium signaling in neutrophils. The objectives of this proposal are to define the molecular pathways that regulate SOCE in neutrophils and to determine how these pathways modulate neutrophil activation during ALI. The central hypotheses are: 1) STIM calcium sensors and ORAI calcium channels cooperate to regulate neutrophil calcium signaling, and 2) calcium-dependent neutrophil activation is critical for induction of ALI. The studies proposed in Aims 1 and 2 will identify the molecular machinery required for SOCE in neutrophils and determine the role of neutrophil calcium signaling in acute lung injury using a unique set of mice with neutrophil specific deletion in Stim or Orai genes. Aim 3 will utilize a novel approach with humanized mice to test these hypotheses in human neutrophils. This project is relevant to the missions of the NIH and NIAID since these studies will elucidate previously unexplored pathways of calcium signaling in neutrophils that may lead to new therapeutic approaches to treat acute lung injury and other inflammatory processes.